CN112771159A - Arginase 1 polypeptides - Google Patents

Abstract

The present invention relates to novel polypeptides derived from arginase 1. The invention also relates to polynucleotides encoding said polypeptides. The invention also relates to compositions comprising said polypeptides and polynucleotides. The invention also relates to uses of the polypeptides, polynucleotides and compositions.

Description

Arginase 1 polypeptides

Technical Field

The present invention relates to novel polypeptides derived from arginase 1. The invention also relates to polynucleotides encoding the polypeptides. The invention also relates to compositions comprising the polypeptides and polynucleotides. The invention also relates to uses of the polypeptides, polynucleotides and compositions.

Background

Arginase is an enzyme that catalyzes the reaction that converts the amino acid L-arginine into L-ornithine and urea. This depletes the arginine microenvironment and results in the suppression of tumor specific cytotoxic T cell responses. Increased arginase activity has been detected in cancer cells of breast, lung, colon, or prostate cancer patients. Mouse macrophages transfected with the rat arginase gene have been shown to promote proliferation of co-cultured tumor cells in vitro and in vivo. Furthermore, induction of arginase expression by macrophages has been shown to increase tumor angiogenesis through polyamine synthesis. Results from the murine lung cancer model indicate the presence of a subpopulation of mature tumor-associated bone marrow cells (myoid cells) that express high levels of arginase. These tumor-associated bone marrow cells deplete extracellular L-arginine, which inhibits antigen-specific proliferation of Tumor Infiltrating Lymphocytes (TILs). In mice, the injection of arginase inhibitors blocked the growth of lung cancer. This demonstrates how induction of arginase expression in tumor cells and tumor-associated bone marrow cells promotes tumor growth by suppressing the anti-tumor immune response (through adverse effects on TIL).

MDSCs (myeloid-derived suppressor cells) inhibit the activation, proliferation and cytotoxicity of effector T cells and natural killer cells, as well as induce Treg differentiation and expansion. Both tumor cells and MDSCs inhibit T cells by manipulating L-arginine metabolism via the enzymes Nitric Oxide Synthase (NOS) and arginase. Many tumors exhibit increased expression of arginase and inducible nos (inos), leading to arginine depletion in the tumor microenvironment. Several studies underscore the importance of this altered tumor arginine metabolism in inhibiting tumor-specific T cell responses, and recently demonstrated that Acute Myeloid Leukemia (AML) BLAST exhibits arginase-dependent ability to inhibit T cell proliferation and hematopoietic stem cells. In addition, arginase and iNOS inhibitors decrease the inhibitory activity of AML.

Disclosure of Invention

The inventors of the present invention have previously identified a region of 50 amino acids of arginase 1, which is a "hot spot" of immunogenicity. This region corresponds to position 161-210 of full-length human arginase 1(SEQ ID NO: 10). This region and peptides derived therefrom are described in WO 2018065563. (note that the terms "arginase 1", "Arg 1", "arginase 1" are used interchangeably herein).

The inventors of the present invention have now identified a particular subset (sub-set) of polypeptides derived from this region as being particularly effective in stimulating an immune response. Thus, the polypeptides of the invention are expected to be particularly effective in stimulating a beneficial immune response against arginase 1 and cells expressing arginase 1. The development of new immunotherapies for cancer requires a full understanding of the molecules involved in pathogenesis as well as the specific proteins recognized by the immune system. In the clinical setting, induction of an arginase-specific immune response, in addition to killing cancer cells, can support an anti-cancer immune response in general by inhibiting the immunosuppressive function of arginase-expressing cells, particularly MDSCs and tumor-associated macrophages (TAMs). Thus, targeting bone marrow dendritic cells (e.g., by immunization with a polypeptide of the invention) would be highly synergistic with other anti-cancer immunotherapies, as cells expressing arginase antagonize the desired effects of other immunotherapeutic approaches.

The present invention provides:

an isolated polypeptide consisting of any one of the following amino acid sequences:

a.ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRL(SEQ ID NO:1)；

b.ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKL(SEQ ID NO:2)；

c.ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSM(SEQ ID NO:3)；

d.ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSM(SEQ ID NO:4)；

e.ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGK(SEQ ID NO:5)；

f.ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKLGIGK(SEQ ID NO:6)。

the invention further provides a polynucleotide encoding a polypeptide of the invention, optionally comprised in a vector.

The invention also provides a composition comprising a polypeptide or polynucleotide of the invention, at least one pharmaceutically acceptable diluent, carrier or preservative, and optionally an adjuvant.

The invention also provides a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a polypeptide, polynucleotide or composition of the invention.

Drawings

FIG. 1 shows the response to a long Arg 1-derived peptide

IFN γ ELISPOT responses to ArgLong, ArgLong2, and ArgLong3 peptides in PBMCs from four Healthy Donors (HD). The histogram represents the mean spot number per well + the standard error of the mean. For each donor, corresponding ELISPOT well images with and without peptide addition are also shown. Experiment at 5X10⁵PBMC/well, triplicate. TNTC-is abundant and cannot be counted. P is less than or equal to 0.05 according to the rule of non-distributed resampling (DFR).

FIG. 2 shows the response to ArgLong2 compared to the 20-mer (20-mer) peptide

In 19 healthy donors (upper panel) and 16 cancer patients (lower panel), Arg was treated_171-190、Arg_181-200、Arg_191-210And the IFN γ ELISPOT response of ArgLong 2. Each dot represents the average number of peptide-specific spots per patient/donor. Responses were calculated by subtracting the average spot count in control wells from the average spot count in peptide-stimulated wells. For healthy donors, experiments were performed at 4.5-5X10⁵PBMC/well, three or two replicates, and for cancer patients, experiments were performed at 2.2-5X10⁵PBMC/well, three or two replicates. The number of responses to ArgLong2 was large and not counted (TNTC) among 6 healthy donors and 2 cancer patients, and was set as>500 spots.

FIG. 3 shows the response of CD4+ and CD8+ T cells to ArgLong2 in vitro (in vitro) and ex vivo (ex vivo)

3A-in vitro response to ArgLong2 in CD4+ (left) and CD8+ (right) T cells in intracellular staining of PBMCs from 2 healthy donors and 1 patient with breast cancer. PBMC were stimulated with ArgLong2 and low dose IL-2 for 1 week prior to ELISPOT assay.

3B-No prior stimulation (BC-breast cancer, MM-malignant melanoma), intracellular staining of PBMCs from 2 cancer patients, ex vivo response to ArgLong2 in CD4+ (left) and CD8+ (right) T cells.

FIG. 4 shows that ArgLong 2-specific CD4+ and CD8+ cells are memory T cells

4A-Ex vivo response to ArgLong2 in 4 Healthy Donors (HD) and 2 cancer patients (BC-breast cancer, MM-malignant melanoma). Experiments were performed at 1-5X10⁵One cell/well was performed in triplicate.

4B-ex vivo IFN γ ELISPOT from sorted CD4+ memory T cells (CD45RO +) from PBMCs of 4 healthy donors and 2 cancer patients (breast cancer and malignant melanoma). Experiments were performed at 1-3X10⁵One cell/well was performed in triplicate.

4C-sorted CD8+ memory T cells (CD45RO +) ex vivo IFN γ ELISPOT from PBMC from 2 healthy donors. Experiments were performed at 1-3X10⁵One cell/well, repeated twice or in a single run. The histogram represents the mean spot count + SEM in peptide-stimulated wells and control wells.

FIG. 5 shows that IL-4 stimulates activation of ArgLong 2-specific T cells

A-IFN γ ELISPOT stimulating ArgLong2 response in PBMCs of melanoma patients for one week by IL-4 and/or IL-2. Unstimulated PBMC were used as controls. The superimposed histogram shows the average number of spots + SEM for the control wells and the peptide-stimulated wells. Experiment was performed at 3X10⁵One cell/well was performed in triplicate. B-ArgLong 2 specific response in PBMCs from 6 Healthy Donors (HD) and 2 cancer patients (BC-breast cancer, MM-malignant melanoma) one week after stimulation with IL-2(120U/ml) or IL-4 (100U/ml). Cells without cytokine stimulation were used as controls. Responses were calculated as the difference between the mean spot counts of peptide-stimulated wells and control wells in ELISPOT. Experiment was performed at 3X10⁵One cell/well, three or two replicates.

FIG. 6 shows the ArgLong2 specific IFN γ immune response generated in vivo following immunization

4 mice were immunized with ArgLong2 peptide and the immune response to the peptide was evaluated 7 days later by IFN γ ELISPOT. Cells from the spleen and draining lymph nodes of a single mouse were subjected to ELISPOT assay. The histogram represents the number of average spots. ELISPOT was performed with and without ArgLong2 peptide, in triplicate. "TNTC" means a large number that cannot be counted. P <0.01 according to the non-distributed resampling method (DFR).

FIG. 7 shows that ArgLong2 immunization induced anti-tumor effects in MC38 colon adenocarcinoma tumor model

On day 0, 0.5X 10⁶One MC38 tumor cell was inoculated subcutaneously in the right flank of 30 mice. Immunization was started on the same day. 15 mice in the treatment group were treated with 100 μ g of ArgLong2 in Montanide emulsion, and 15 mice in the control group were treated with H in Montanide emulsion₂And (4) O treatment. According to the analysis of the mixing effect, p is 0.030.

FIG. 8 shows that ArgLong2 immunization induces anti-tumor effects in B16F10 melanoma tumor model

On day 0, 0.5X 10⁶One B16F10 tumor cell was inoculated subcutaneously in the right flank of 30 mice. Immunization was started on the same day. 15 mice in the treatment group were treated with 100 μ g of ArgLong2 in Montanide emulsion, and 15 mice in the control group were treated with H in Montanide emulsion₂And (4) O treatment. After three immunizations, treatment was stopped. When the tumor volume exceeds 864mm³Mice were euthanized at time.

FIG. 9 shows that ArgLong 2-specific T cell clones recognize THP-1 cells expressing arginase 1

A and B-when arginase 1 expression is induced by Th2 cytokine, the ArgLong2 specific CD4T cell clone recognizes THP-1 cells, which is quantified by intracellular cytokine staining. C-qPCR data show that IL-13 pre-stimulation of THP-1 cells induces arginase 1 expression.

FIG. 10 shows that cytokine stimulation alone in vitro is sufficient to enhance T cell response to the ArgLong2 epitope

PBMCs were treated with either Th2 cytokine (IL-4/IL-13) or ArgLong2 peptide inducing arginase 1 for 7 days, and ArgLong2 specific T cells were then quantified in vitro by IFN γ ELISPOT.

Brief description of the sequences

1-9 are each the amino acid sequence of a polypeptide derived from the region corresponding to position 161-210 of full-length human arginase 1 or murine arginase 1.

SEQ ID NOS 10 and 11 are amino acid sequences of full-length human arginase 1 and mouse arginase 1.

12 is the amino acid sequence of the region corresponding to position 161-210 of full-length human arginase 1.

13 is the amino acid sequence of the region corresponding to position 161-210 of full-length murine arginase 1.

Detailed Description

It is understood that different applications of the disclosed products and methods may be varied to suit particular needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides" and the like.

"polypeptide" as used herein refers in its broadest sense to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences, as well as longer polypeptides and proteins. The term "amino acid" as used herein refers to natural and/or unnatural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptidomimetics.

The terms "patient" and "subject" are used interchangeably and generally refer to a human.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

The inventors of the present invention have previously identified a region of 50 amino acids of human and murine arginase 1, which is a "hot spot" for immunogenicity. This region corresponds to position 161-210 of full-length human arginase 1(SEQ ID NO:10) or full-length murine arginase 1(SEQ ID NO: 11). This region and peptide fragments derived therefrom are described in WO 2018065563. The inventors of the present invention have now found that polypeptides consisting of a particular contiguous amino acid sequence of the same region are unexpectedly immunogenic with respect to other amino acid sequences from that region.

By "immunogenic" herein is meant that the polypeptide is capable of eliciting an immune response to the arginase 1 protein, preferably when said protein is present in or on cells expressing the arginase 1 protein. In other words, the polypeptide may be described as immunogenic for arginase 1. Alternatively the polypeptide may be described as an immunogenic fragment of arginase 1. The immune response is preferably a T cell response. After administration of the polypeptide to at least one individual (or in a sample obtained from the individual), an immune response may be detected in the individual (or the sample).

Any suitable method, including in vitro methods, can be used to identify a polypeptide as immunogenic. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics:

(i) which is capable of inducing IFN-gamma producing cells in a PBL population of a healthy subject and/or a cancer patient, and/or by an ELISPOT assay

(ii) Which is capable of detecting in situ CTLs reactive with arginase 1 in a tumor tissue sample;

and/or

(iii) Which is capable of inducing the growth of specific T cells in vitro.

The following examples section also describes methods suitable for determining whether a polypeptide has immunogenic activity.

The polypeptide of the invention may comprise:

the amino acid sequence corresponding to position 169-206 of full-length human arginase 1. This polypeptide may be referred to herein as ArgLong 2. The sequence is given as SEQ ID NO 1.

An amino acid sequence corresponding to position 169-206 of full-length murine arginase 1. This polypeptide may be referred to herein as magglong 2. The sequence is provided as SEQ ID NO 2.

The amino acid sequence corresponding to position 169-200 of full-length human arginase 1. This polypeptide may be referred to herein as ArgLong 3. The sequence is provided as SEQ ID NO 3.

An amino acid sequence corresponding to position 169-200 of full-length murine arginase 1. This polypeptide may be referred to herein as magglong 3. The sequence is provided as SEQ ID NO 4.

An amino acid sequence corresponding to position 169-210 of full-length human arginase 1. This polypeptide may be referred to herein as ArgLong. The sequence is provided as SEQ ID NO 5.

An amino acid sequence corresponding to position 169-210 of full-length murine arginase 1. The polypeptide may be referred to herein as a magglong. The sequence is provided as SEQ ID NO 6.

The polypeptide preferably consists of the amino acid sequence corresponding to position 169-206 of the full-length human or murine arginase 1, i.e.it consists of the amino acid sequence of SEQ ID NO 1 or 2. That is, the polypeptide is preferably ArgLong2 or maglong 2.

The polypeptide most preferably consists of the amino acid sequence corresponding to position 169-206 of full-length human arginase 1, i.e., it consists of the amino acid sequence of SEQ ID NO: 1. That is, the polypeptide is preferably ArgLong 2.

In any of the polypeptides described herein, the amino acid sequence may be modified by 1, 2, 3, 4 or 5 (up to 5) additions, deletions or substitutions, provided that the polypeptide having the modified sequence exhibits the same or increased immunogenicity to arginase 1 as compared to the polypeptide having the unmodified sequence. By "identical" it is understood that the polypeptide of modified sequence does not exhibit significantly reduced immunogenicity for arginase 1 as compared to the polypeptide of unmodified sequence. Any comparison of immunogenicity between sequences was performed using the same assay. Unless otherwise indicated, modifications of the polypeptide sequence are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties, or similar side chain volumes. The introduced amino acid may have a polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge similar to that of the amino acid it replaces. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid to replace an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected based on the properties of the 20 major amino acids defined in table a1 below. If the amino acids have similar polarity, this can be determined by reference to the hydrophilicity scale (hydropathiy) of the amino acid side chain in Table A2.

TABLE A1 chemical Properties of amino acids

TABLE A2-level of hydrophilicity

In any of the polypeptides disclosed herein, any one or more of the following modifications may be made to improve its physiochemical properties (e.g., stability) provided that the polypeptide exhibits the same or increased immunogenicity for arginase 1 as compared to a polypeptide having an unmodified sequence:

a) replacement of the C-terminal amino acid by the corresponding amide (increased resistance to carboxypeptidase)

b) Replacement of the N-terminal amino acid with the corresponding acylated amino acid (which may increase resistance to aminopeptidases);

c) replacement of one or more amino acids with the corresponding methylated amino acid (which may increase proteolytic resistance);

d) one or more amino acids are replaced with the corresponding amino acid in the D-configuration (which may increase proteolytic resistance).

For modifications of class (c) and (D), a preferred example is the modification of the Tyr residue in the position corresponding to position 29 of SEQ ID NO:1 (e.g.replacement with N-methyl (Tyr) or replacement with the D configuration Tyr). This is because Tyr occurs just after the Lys residue at position 28 and is therefore located at the potential site of proteolysis by trypsin-like proteases (which typically cleave after Lys).

Any of the polypeptides disclosed herein may be linked at the N-and/or C-terminus with at least one additional moiety to improve solubility, stability and/or facilitate preparation/isolation, provided that the polypeptide exhibits the same or increased immunogenicity for arginase 1 as compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequence KK, KR or RR may be added at the N-terminus and/or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

The polypeptides disclosed herein may be prepared by any suitable method. For example, the polypeptide can be synthesized directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry, or by solution phase peptide synthesis. Alternatively, the polypeptide may be prepared by transforming a cell (typically a bacterial cell) with a nucleic acid molecule or vector encoding the polypeptide. The invention provides nucleic acid molecules and vectors encoding the polypeptides of the invention. The invention also provides a host cell comprising the nucleic acid or vector.

The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolation of any sequence, RNA, nucleic acid probes, and primers. The polynucleotides of the invention may be provided in isolated or substantially isolated form. Substantially isolated means that the polypeptide can be substantially, but not completely, isolated from any surrounding medium. The polynucleotides may be mixed with a carrier or diluent that does not interfere with their intended use, and are still considered substantially isolated. A nucleic acid sequence "encoding" a polypeptide of choice is a nucleic acid molecule that is transcribed (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vivo when placed under the control of appropriate regulatory sequences, for example in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the present invention, such nucleic acid sequences may include, but are not limited to, cDNA from a virus, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence.

Polynucleotides can be synthesized according to methods well known in the art, as described, for example, in Sambrook et al (1989, Molecular Cloning-a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecule of the invention may be provided in the form of an expression cassette (expression cassette) comprising control sequences operably linked to an insertion sequence, thereby allowing the expression of the polypeptide of the invention in vivo. These expression cassettes are in turn typically provided within a vector (e.g., a plasmid or recombinant viral vector). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the present invention may be administered to a host subject. Preferably, the polynucleotide is prepared and/or administered using a genetic vector. Suitable vectors may be any vector capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptide of the invention.

Accordingly, the present invention includes expression vectors comprising such polynucleotide sequences. Such expression vectors are constructed in conventional manner in the field of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiation codons (initiators), promoters, enhancers and other elements (such as polyadenylation signals which may be necessary) and which are placed in the correct orientation to allow expression of the peptides of the invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, the inventors refer to Sambrook et al.

The invention also includes cells that have been modified to express the polypeptides of the invention. Such cells typically include prokaryotic cells, such as bacterial cells (e.g., e. Such cells can be cultured using conventional methods to produce the polypeptides of the invention.

The polypeptides of the invention may be in a substantially isolated form. It may be mixed with carriers, preservatives or diluents (discussed below) that do not interfere with the intended use, and/or with adjuvants (also discussed below), and still be considered substantially separate. It may also be in a substantially purified form, in which case it will generally comprise at least 90% (e.g. at least 95%, 98% or 99%) of the protein in the formulation.

Compositions comprising polypeptides or polynucleotides

In another aspect, the invention provides a composition comprising a polypeptide of the invention. The invention also provides compositions comprising polynucleotides encoding the polypeptides of the invention. For example, the invention provides compositions comprising one or more polypeptides of the invention, and at least one pharmaceutically acceptable carrier, preservative or excipient. Alternatively, the invention provides a composition comprising one or more polynucleotides encoding a polypeptide of the invention, and at least one pharmaceutically acceptable carrier, preservative or excipient. The carriers, preservatives and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the subject to which the composition is administered. Typically, all components and the final composition are sterile and pyrogen-free. The composition may be a pharmaceutical composition. The composition may preferably comprise an adjuvant.

An adjuvant is any substance that when mixed into a composition will increase or otherwise alter the immune response elicited by the composition. An adjuvant is defined broadly as a substance that promotes an immune response. Adjuvants may also preferably have a depot effect, i.e. they also provide a slow and sustained release of the active agent from the site of administration. A general discussion of adjuvants is provided at pages 61-63 of Goding, Monoclonal Antibodies: Principles & Practice (second edition, 1986).

The adjuvant may be selected from: AlK (SO)₄)₂，AlNa(SO₄)₂，AlNH₄(SO₄) Silicon dioxide, alum, Al (OH)₃，Ca₃(PO₄)₂Kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-desmethylmuramyl (nonnurmyl) -L-alanyl-D-isoglutamine (CGP 11687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'2' -dipalmitoyl-sn-trioxy-3-hydroxyphosphooxy) -ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL + TDM + CWS) in 2% squalene/Tween-80.. emulsion, lipopolysaccharides and various derivatives thereof, including lipid A, Freund's Complete Adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g. poly IC and poly AU acids), D waxes from Mycobacterium tuberculosis (Mycobacterium tuberculosis), substances present in Corynebacterium parvum, Bordetella pertussis (Bordetella pertussis) and members of the Brucella genus (Brucella), Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21. Various saponin (saponin) extracts have also been suggested for use as adjuvants in immunogenic compositions. Granulocyte-macrophage colony stimulating factor (GM-CSF) may also be used as an adjuvant.

Preferred adjuvants for use in the present invention include oil/surfactant based adjuvants such as Montanide adjuvant (available from Seppic, belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA-based adjuvants, such as adjuvants comprising CpG oligonucleotide sequences. Other preferred adjuvants are viral dsRNA based adjuvants such as poly I: C. GM-CSF and Imidazociline are also examples of preferred adjuvants.

Most preferably, the adjuvant is Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

In Goding, Monoclonal Antibodies: Principles & Practice (second edition, 1986), pages 61-63, it is also described that coupling to immunogenic carriers is recommended when the antigen of interest has a small molecular weight or is poorly immunogenic. Thus, the polypeptides of the invention may be coupled to a carrier. The carrier may be present independently of the adjuvant. The function of the carrier can be, for example, to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, to confer stability, to enhance biological activity, or to increase serum half-life. In addition, the carrier may aid in the presentation of the polypeptide or fragment thereof to the T cell. Thus, in the composition, the polypeptide may be associated with a carrier (such as those described below) (an associate).

The carrier may be any suitable carrier known to those skilled in the art, such as a protein or an antigen presenting cell, such as a Dendritic Cell (DC). The carrier protein includes keyhole limpet hemocyanin (keyhole limpet hemocyanin), serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. Alternatively, the carrier protein may be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier may be dextran, such as agarose. The carrier must be physiologically acceptable and safe for humans.

If the composition comprises an excipient, it must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in the excipients. These excipients and auxiliary substances are usually pharmaceutical agents (pharmacological agents) which do not elicit an immune response in the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. It may also contain pharmaceutically acceptable salts, such as inorganic acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; organic acid salts such as acetate, propionate, malonate, benzoate, and the like. For a detailed discussion of pharmaceutically acceptable excipients, carriers (vehicles) and auxiliary substances, see Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991).

Formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemistry and methods, all of which are readily available to those skilled in the art. The composition may be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged or sold in unit dosage form (e.g., in ampoules) or in multi-dose containers optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations. In one embodiment of the composition, the active ingredient is provided in a dry form (e.g., powder or granules) for reconstitution with a suitable solvent (vehicle) (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oleaginous suspensions or solutions. The suspension or solution may be formulated according to known prior art and may contain, in addition to the active ingredient, other ingredients such as adjuvants, excipients and auxiliary substances as described herein. Such sterile injectable preparations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, for example, water or 1, 3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and non-volatile oils (e.g., synthetic mono-or diglycerides). Other compositions that may be used include compositions comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. Alternatively, the active ingredient of the composition may be encapsulated (encapsulated), absorbed into a particulate carrier or associated with a particulate carrier (adjuvant). Suitable particulate carriers include PLG particulates derived from polymethylmethacrylate polymers, as well as from poly (lactide) and poly (lactide-co-glycolide). See, e.g., Jeffery et al (1993) pharm. Res.10: 362-. Other particulate systems and polymers may also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

Application method

The polypeptides, polynucleotides, or compositions of the invention may be used in methods of treating or preventing a disease or disorder in a subject. The polypeptides, polynucleotides, or compositions of the invention may be used in the manufacture of a medicament for use in a method of treating or preventing a disease or disorder in a subject. The method comprises administering the polypeptide, the polynucleotide, or the composition to the subject. A therapeutically or prophylactically effective amount of the polypeptide, the polynucleotide, or the composition can be administered to a subject in need thereof.

The disease or condition may be characterized, at least in part, by inappropriate or excessive immunosuppressive function of arginase 1. The disease or disorder may be cancer, preferably cancer that expresses arginase 1 and/or is associated with inappropriate or excessive immunosuppressive function of arginase 1. The cancer may be breast, lung, colon or prostate cancer, or may be a leukemia, preferably Acute Myeloid Leukemia (AML), or may be a melanoma.

The methods may comprise administering additional cancer therapies simultaneously or sequentially. The additional cancer therapy may be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiation therapy, immunostimulatory substances, gene therapy, or antibodies.

The antibody may be abamectin (Abagovamab), Abciximab (Abciximab), Abciximab (Actoxumab), Adalimumab (Adalilimumab), Adalimumab (Adecatuzumab), Alemtuzumab (Adecatuzumab), Aframomumab (Afelimomab), afuzumab (Afituzumab), Periplexizumab (Alizezumab), ALD518, Alemtuzumab (Alemtuzumab), alelimumab (Alitumumab), Aituzumab (Alitumumab), pentostatin Actuzumab (Altuzumab pentamumab), Amaximab (Amatuzumab), Analimumab (Anatomumab), Antuzumab (Anatumumab), Anrutuzumab (Anrukinumab), Apolizumab (Apolizumab), Acitumumab (Artuzumab), Asaguizumab (Asagovab), Abxizumab (Abelizumab), Abelizumab (Ab), Abelizumab (Betuzumab), Abelizumab (Betuzumab (Abelizumab), Abelizumab (Abelizumab), Abelizumab (Abelix), Abelix (Abelix, Bevacizumab (besilezumab), Bevacizumab (Bevacizumab), Bevacizumab (Bezlotoxumab), bizumab (Biciromab), Bimagrumab (Bimagrumab), mobitumumab (Bivatuzumab mertansine), bonatumumab (blinatumumab), Blosozumab (rituximab (Brentuximab vedotine), brikinumab (briakininumab), Brodalumab (Brodalumab), Canakinumab (Canakinumab), mototuzumab (canum), cantitamin (cantitan), carpuzumab (caput), carpuzumab (carpuzumab), carpuzumab (catuzumab), carpuzumab (carpuzumab), carpuzumab (caput), carpuzumab coupling (caput), carpuzumab (cituzumab), cituzumab (civetuzumab), civetutab (civetutab), Cetuximab (cituzumab (civetutab), civetutab (cituzumab), civetutab (civetutab), civetutab (civetutab, ci, Kang tuzumab (Conatumumab), conximab (Concizumab), clenbuterol (Crenezumab), CR6261, Dazizumab (Dacetuzumab), Dalizumab (Dacetizumab), Dalizumab (Daclizumab), Daltuzumab (Daltuzumab), Dalamumab (Daratumumab), Deximab (Demcizumab), Desuzumab (Denosumab), Demomab (Detumomab), Dermomab (Detumomab), Dorlimomaaritox (Dorlimomaaritox), Drozimab (Drozimab), Duritumumab (Duligoumab), Dougumab (Dupimelimab), Duximab (Duxizumab (Duitsiguzumab), Esomeximab (Ecromeximab), Ekulizumab (Esomezumab), Edobaumib (Edomolimumab), Edomomazumab (Edomomazumab), Esomemazumab (Evomazumab), Evomazumab (Entrovazumab), Evomazumab (Entuzumab), Evomazumab (Entroxib (Evomazumab), Evomazomab (Enliomazokub), Evomazomab (Evomazokub), Evomazokumazomab (Evomazoku, Epratuzumab (Epratuzumab), Erlizumab (Erlizumab), Eimazemazumab (Ertumaxomab), Eitalizumab (Etaracizumab), Etricizumab (Etrolizumab), Evouzumab (Evolvulumab), Evomazumab (Exbivirumab), Fanolesomab, Faramomumab (Faralimomab), Falutuzumab (Farleuzumab), Fasinumab (Fajinumab), FBTA05, Falvizumab (Felvizumab), Fizanitumumab (Fezakinumab), Noctuzumab (Filattuzumab), Figituzumab (Figituzumab), Flankitumumab (Flankitumumab), Fultuzumab (Flankitumumab), Furtuzumab (Flankuzumab), Furtuzumab (Futamumab), Fujiuzumab (Frankuzumab), Gamituzumab (Gamituzumab), Fujituzumab (Fujiuzumab), Fujiuzumab (Fujiuzumab), Gamituzumab (Gegimuzumab), Fujiuzumab (Gegimuzumab), Gajiuzumab (Gegimuzumab), Fujiuzumab (Gegimuzumab), Gegimuzumab (Gegimutab), Gejikumazoutab (Gejikumazoutab), Gejikumazokumazokumazokumazokumazokumab (Gejikumazokumab), Gejikumazokumazokumab), Gejikumab, Gemtuzumab ozogamicin (Girentuzimab), Vigrezumab (Glempatumumab vedotin), Golimumab (Golomimab), high-density abciximab (Gomiliximab), GS6624, Ibalizumab (Ibasizumab), Ibritumomab tiuximab conjugate (Ibritumumab tiuxetan), Ibritumomab (Ibrucuumab), agovacizumab (Igovamab), Infliximab (Imciromab), Engatuzumab (Imgatuzumab), Icalamus (Inclacumab), Intuximab revtanine (Inatuzumab), Infliximab (Intuximab ravtansine), Infliximab (Infliximab), Intetumumab (Intitumumab), inolimumab (Indomumab), Infliximab), Lelizumab (Lelizumab), Lelizumab (Illinumumab), Lelizumab (Illinamumab), Infliximab (Izemazumab), Lelizumab (Izerumab (Izemazumab), Lelizumab (Illinumab), Lelizumab (Izerumumab), Lelizumab (Izerumab), Lelizumab (Izerumumab), Lelizumab (Izerumab), Lelizumab), and Lelizumab (Izelizumab), and Lelizumab) or (Izelizumab), and a, Ribavirin mab (Libivirumab), Ligelizumab, Lintuzumab (Lintuzumab), Lirilumab, ludwizumab (lucatelimumab), lumiximab, mapitumumab (Lodelcizumab), molovatuzumab (lorvovatuzumab mertansine), lucatumab (lucatumab), lumiximab, mapitumumab (mapitumumab), massecuzumab (malimumab), mettiumumab (Metelimumab), miraculin (maitsumomab), milmomab (Minretumomab), mitumumab (Mitumomab), magalizumab (moguamtuzumab), molamulizumab (molamulimumab), mupivoximab (momab), muvirtuzumab (momab), netuzumab (nevatuzumab), netuzumab (minremozumab), mituzumab (mitutuzumab), magamumatuzumab (mituzumab), magalizumab (momab), momutamtuzumab (natuzumab), netovatuzumab (naoxutamab), naitumomab (nautamab (nakamtuzumab), nautamab (nautamab), nautamab (nautamate), nautamab (nautakutuzumab), nautab), nakatuzumab-84), nakatuzumab (nakayamautab), amatuzumab-kayamatuzumab (nakayamaku, Nerrimumab (Nerelimomab), nesivazumab (nesvacuumab), Nimotuzumab (Nimotuzumab), Nivolumab (Nivolumab), Nimotuzumab (Nivolumab), minomomab-meperizumab conjugate (nofetumumab merpentan), atozumab (Obinutuzumab), ocaluzumab (ocartuzumab), ocreliximab (Ocrelizumab), Ocrelizumab (Ocrelizumab), ondumumab (odumumab), ofazumab (ovazezumab), ocurizumab (odonumab), otuzumab (ovatuzumab) conjugate (scattering optometriab), agogozumab (orizumab), omamotizumab (orimotizumab), nonatezumab (paratuzumab), atrazumab-monatin conjugate (scattering optomab), agogozumab (orizumab), orizumab (origozumab), orizumab (orimotizumab), nonatezumab (parkizumab), otelizumab (oclavumab), otelizumab (oclavirb), paclizumab (oclavumab), paclizumab (oclavizumab), paclizumab (oclavumab), paclizumab (ocrelib), paclobulizumab (Ocrelizumab), paclobulizumab (Ocrelizumab), paclobutanil (ocrelib), paclobutanil (Ocrelizumab), paclobutanil (Ocrelizumab), paclobutanil (Ocrelizumab), paclobutanil (ocreli, Pertuzumab (Patritumumab), panitumumab (Pemtumomab), Pilatumab (Perakizumab), Pertuzumab (Pertuzumab), pexizumab (Pexizumab), PIRILIzumab (Pidilizumab), Vipinitumumab (Pinatuzumab vedotin), Ponteuzumab (Pintumomab), Prugumab (placuluumab), Potuzumab (Polatuzumab vedotin), Poncizumab (Ponezumab), Pruliximab (Priliximab), Pritoxamab (Pritoxamab), Pritumumab (Pritumumab), PRO 140, Quinizumab (Quililizumab), Ratuzumab (Ratumomab), Ratuzumab (Ratuzumab), Ratuzumab (Rituzumab), Ratuzumab (Rituzumab (Rotuzumab), Ratuzumab (Rotuzumab), Ratuzumab (Rotuzumab), Ratuzumab (Rotuzumab) and Ratuzumab (Rotuzumab) in, Rovelizumab (Rovelizumab), lucuzumab (Ruplizumab), samurizumab (Samalizumab), Sarilumab, Sarimumab-pentosan conjugate (Satemab pendentide), Securizumab (Secukinumab), Sedrizumab (Seribantumab), Setuximab (Setoxiximab), Sevemab (Sevirumab), Sibrolizumab (Sibrolizumab), Sifalumab (Sifalimumab), Securizumab (Sifalamumab), Setuximab (Siteuximab), Situzumab (Situzumab), Situzumab (Simtuzumab), Ceprizumab (Siplizumab), Siplizumab (Siplizumab), Sirukuumab (Sirukumab), Sulezumab (Solazezumab), Solulizumab (Solidab), Suvelumab (Suvelumab), Suveluzumab (Suveluzumab), Suvelumab (Suveltuzumab), Suveltuzumab (Suvelutab), Suvelutab (Tavelutab), Suveluzumab (Suvelutab (Taveluzumab), Suvelutab (Suveluzumab), Suvelutab (Suvelutab), Suveluzumab (Suveluzumab), Suveltuzumab), Suveluzumab (Suveltuzumab), Suvellus (Suvella), Suveltuvella), Suveltevella (Suveltevella), Suvelteveltuzumab), Suveluzumab), Suvella (Suveluzumab), Suvella (Suvelte, Attentiomab (Telimomab), tenemumab (tentuzumab), tenetuzumab (tentuzumab), tenenximab (Teneliximab), tenelizumab (Teplizumab), tetrapropylumab (teprotuzumab), TGN1412, tenexizumab (ticimumab) (═ Tremelimumab), tenerazumab (tiltrakizumab), tegafuzumab (Tigatuzumab), TNX-650, tolizumab (Tocilizumab) ((atelizumab)), tolazazumab (Toralizumab), Tositumomab (Tositumomab), trolizumab (trakinumab), Trastuzumab (Trastuzumab), TRBS07, Trastuzumab (degalizumab), tremetuzumab (tretuzumab), tretuzumab-tuzumab (valtuzumab), valtuzumab (valtuzumab), coupling (valtuzumab), valtuzumab (valtuzumab), valtuzumab (valtuzumab), coupling (valtuzumab) and (valtuhut, Vepamumab (Vepalimomab), vessenmab (Vesencumab), vesizumab (Visilizumab), volvacizumab (Volociximab), volzeitumumab-mazetin conjugate (voretuzumab), volitumumab (volitumumab), Zalutumumab (Zalutumumab), zaolimumab (Zanolimumab), Zatuximab, zilamumab (ziralamumab), or adolimumab (zoimox).

Preferred antibodies include natalizumab, vedolizumab, belimumab, asexumab (Atacicept), Alefacept (Alefacept), oriliximab, tilizumab, rituximab, ofatumumab, oxecoximab, eprinolizumab, epratuzumab, eculizumab (Abatacept), eculizumab, omalizumab, canazinumab, merizumab (Meplizumab), rayleigh mab, tositumumab, ursinumab, brimonizumab, briyamine, Etanercept (Etanercept), Inlfliximab, adalimumab, certlizumab, golimumab, trastuzumab, gemtuzumab, oxepirubicin, ibritumomab, tositumomab, cetuximab, valtuzumab, parvub, sumitumomab, rituximab, and rituximab.

Particularly preferred antibodies that can be used in the methods of the invention include: darunavir, nivolumab, parbolizumab (pembrolizumab), avizumab (avelumab), rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, parlimumab, tositumomab, and ofatumumab. Especially preferred is daratumab.

Other cancer therapies are selected from: b12(Actimide), Azacitidine (Azacitidine), Azathioprine (Azathioprine), Bleomycin (Bleomycin), Carboplatin (Carboplatin), Capecitabine (Capecitabine), Cisplatin (Cisplatin), Chlorambucil (Chlorambucil), Cyclophosphamide (Cyclophosphamide), Cytarabine (Cytarabine), daunorubicin (Dauno-rubicin), Docetaxel (Docetaxel), deoxyfluorouridine (Doxifluridine), Doxorubicin (Doxorubicin), Epirubicin (Epiribin), Etoposide (Etoposide), Fludarabine (Fluudarebine), fluorouracil (Fluor-ouracil), Gemcitabine (Gemcitabine), Hydroxyurea (Hydroxyurea), Idarubicin (Illicin), Irinotecan (Idaxanthine), Paclitaxel (Melothalamine), mellituramine (Melothricin), mellithamine (Melphalan), mellitorine (Melphalan), Melphalan (Melphalan), Melphalan (Melphalan), Melphalan (Melphalan), Melphalan (Melphalan), Melphalan (Mel, Temozolomide (Temozolomide), Teniposide (Teniposide), Thioguanine (Thioguanine), Valrubicin (Valrubicin), Vinblastine (Vinblastine), Vincristine (vincrisine), Vindesine (Vindesine), and Vinorelbine (Vinorelbine).

The polypeptides or compositions of the invention may also be used in methods of stimulating arginase 1-specific T cells (e.g., CD4T cells and CD8T cells) comprising contacting the cells with the polypeptides or compositions. The method may be performed ex vivo. The cells may be present in a sample obtained from a healthy subject or a cancer patient, for example in a tumor sample.

The invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope of the invention. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Example 1

Materials and methods

Patient material

Using density gradient separation method (Lymphoprep)^TM(STEMCELL Technologies)) PBMCs from healthy donors were isolated and cryopreserved in FBS supplemented with 10% DMSO at-150 ℃. PBMCs from cancer patients are isolated from blood samples at least four weeks after termination of any anti-cancer therapy. The protocol was approved by the scientific ethics committee of the first city region of denmark and was performed according to the provisions of the declaration of helsinki. Written informed consent was obtained from the patients prior to study entry.

Peptides

Peptides were synthesized by standard methods and provided in the form of solutions in DMSO to achieve a stock concentration of 10 mM. The sequences of the peptides used in these experiments are shown in their entirety below in alignment with each other and are also listed in the section entitled "sequences". The peptides are described by SEQ ID NO, by name, or by reference to the start and stop positions of the respective peptide sequence within the full length sequence of arginase 1. Each description may be used interchangeably. For example, the peptide of SEQ ID NO:1 can either be referred to by the name ArgLong2 or can be referred to as Arg 169-206 (giving start position 169 and stop position 206). The intended mention in each case is clear on the basis of the context.

The longer peptide sequence:

20-mer peptide:

ELISPOT assay

For in vitro ELISPOT, PBMCs from cancer patients and healthy donors were treated with 20mM arginase 1-derived peptide and 120U/ml IL-2 pulse (pulse) in 24-well plates for 7 days before being used in ELISPOT assays. Cells were placed in 96-well nitrocellulose ELISPOT plates (MultiScreen MAIP N45; Millipore) pre-coated with IFN γ capture antibody (Mabtech). Arginase peptide was added to a final concentration of 5 μ M and the plates were incubated at 37 ℃ for 14-16 hours. After incubation, cells were washed and a second biotinylated Ab (Mabtech, Cat. No. 3420-6-1000) was added at room temperature for 2 hours. Unbound secondary antibodies were washed away and streptavidin-conjugated Alkaline Phosphatase (AP) (Mabtech, cat # 3310-10) was added at room temperature for 1 hour. Unbound conjugating enzyme was washed away and the assay developed by addition of BCIP/NBT substrate (Mabtech, catalog 3650-10). The developed ELISPOT plates were analyzed on a CTL ImmunoSpot S6Ultimate-V analyzer using ImmunoSpot software V5.1. Responses were reported as the difference between the average number of spots in wells stimulated with arginase 1 and wells without added peptide.

To examine IL-4 induced Arg1 responses, PBMCs were stimulated with IL-4(100 or 50U/ml) and/or IL-2(120 or 60U/ml) for one week prior to use in the ELISPOT assay described above.

Intracellular staining

In BD GolgiPlug^TMIntracellular staining of cell cultures was performed 5 hours after stimulation of PBMCs with arginase-derived peptides in the presence (added after the first hour of peptide stimulation). Stimulated cells were stained with fluorescently labeled antibodies for surface labeling (CD3, CD4, CD8) and then permeabilized according to the manufacturer's instructions using an immobilization/permeabilization and permeabilization buffer (eBioscience, catalog No. 00-5123-43). The permeabilized cells were then stained with fluorochrome-labeled antibodies to IFN γ and TNF α. In FACSCANTO^TMII (BD biosciences) flow cytometry analysis was performed. According to the manufacturer's instructions, the antibodies used: IFN γ -APC (Cat. No. 341117), TNF α -455BV421 (Cat. No. 562783), CD4-FITC (Cat. No. 347413), CD8-PerCP (Cat. No. 345774), CD3-APC-H7 (Cat. No. 560275) (all from BD Biosciences), dead cell dye-FVS 510(564406, BD Biosciences).

Memory T cell sorting

CD4+ and CD8+ memory T cells were sorted from PBMC samples of freshly thawed healthy donors or cancer patients using a magnetic bead sorting kit: memory CD4+ T cell isolation kits, human (catalog No. 130-. The purity of the isolated cells was assessed by staining for CD4-FITC, CD8-PerCP, CD45RO-PE (all from BD Biosciences), using LIVE/DEAD^TMFixable near-infrared dead cell staining kit (Invitrogen)^TMThermo fisher Scientific) stained dead cells.

Results

Arginase 1 peptide length determines the efficiency of T cell stimulation

Based on the previously identified Arg1 hot spot region 50 amino acids long at position 161-210 of arginase 1, three different peptides covering the major part of this region were selected, which also excluded the cysteine at position 168 of arginase 1 (expected to improve manufacturability, solubility and stability). The three peptides are: 42-mer ArgLong (position 169-210), 38-mer ArgLong2 (position 169-206) and 32-mer ArgLong3 (position 169-200).

To test whether these peptides could be used to identify arginase 1 responses PBMCs from 6 healthy donors were screened for response in IFN γ ELISPOT. PBMC were stimulated with ArgLong2 peptide and low dose IL-2 for 1 week prior to ELISPOT. Despite the sequence similarity, the ArgLong2 peptide appears to be superior in stimulating T cell responses in IFN γ ELISPOT. As shown in fig. 1, a high response to the ArgLong2 peptide was observed in 4 of 6 donors, while there was a low or no response to the ArgLong peptide and the ArgLong3 peptide.

ArgLong2 is only 4 amino acids shorter than ArgLong and 6 amino acids longer than ArgLong 3. This suggests that peptide length and sequence may play a key role in ensuring optimal processing and presentation of arginase peptides. Thus, it may influence the ability of this peptide to activate Arg 1-specific T cells.

To determine whether the ArgLong2 peptide is comparable to the single 20-mer peptide covering the same sequence described previously (see WO2018065563 examples 1, 2 and 3), PBMCs from 19 healthy donors and 16 cancer patients (8 melanoma, 6 multiple myeloma, 1 breast cancer and 1 renal cell carcinoma) were screened for response against three 20-mer peptides and 38-mer ArgLong 2. ArgLong2 produced the most responses in cancer patients and healthy donors compared to all three 20-mer peptides (see figure 2). Strong responses to ArgLong2 were observed in 14 out of 19 healthy donors and 8 out of 16 cancer patients. Responses to the 20-mer peptide were also observed, although these responses were less and lower. To summarize: arg_171-190The response, Arg, was shown in 3 healthy donors and 3 cancer patients_181-200The response, Arg, was shown in 4 healthy donors and 6 cancer patients_191-210Responses were shown in 7 healthy donors and 3 cancer patients.

CD4+ and CD8+ responses to arginase 1

The previously described T cell response against the 20-mer Arg1 peptide has been shown to be predominantly CD4+ T cell responses in cancer patients and healthy donors (see WO2018065563 example 2). Since the ArgLong2 peptide appears to be more efficiently processed and subsequently recognized by T cells, the type of T cell response to ArgLong2 was investigated by intracellular staining.

PBMCs from two healthy donors (HD384, HD400) and one breast cancer patient (BD30) that previously showed strong response to ArgLong2 in ELISPOT were tested by intracellular staining after one week of in vitro stimulation with ArgLong2 and low dose IL-2. Notably, CD4+ and CD8+ T cell responses to ArgLong2 peptide were identified in cancer patients and healthy donors. CD4+ responses (left panel of fig. 3A) and CD8+ responses (right panel of fig. 3A) were detected in PBMCs of one healthy donor and breast cancer patient. Other healthy donors showed only CD8+ T cell responses.

Notably, detectable CD4+ and CD8+ responses to ArgLong2 were also observed in intracellular staining of PBMCs without prior peptide stimulation. PBMCs from 2 cancer patients (1 malignant melanoma (MM27) and 1 breast cancer (BC30)) were thawed and tested directly for response to ArgLong2 peptide in ex vivo intracellular staining. The inventors were able to detect ex vivo CD4+ responses in PBMCs from 2 cancer patients (fig. 3B, left) and ex vivo CD8+ responses in PBMCs from breast cancer patients (fig. 3B, right).

Memory CD4+ and CD8+ responses to arginase 1

A strong response against ArgLong2 indicates that the frequency of Arg1 specific T cells could be more commonly detected in PBMC without any prior peptide stimulation. PBMCs from 4 healthy donors and 2 cancer patients (1 breast cancer and 1 malignant melanoma) that showed strong responses in the in vitro ELISPOT were tested directly for response to ArgLong2 peptide in ex vivo IFN γ ELISPOT. The inventors found a significant spontaneous response in all 6 donors (see fig. 4A).

There was a strong spontaneous ex vivo response to ArgLong2 peptide in PBMCs of healthy donors and cancer patients, indicating that arginase 1-specific cells are a natural part of the immune system. To test CD4+ and CD8+ memory T cells were selected from PBMCs of cancer patients and healthy donors who showed strong spontaneous ex vivo immune responses. CD4+ or CD8+ memory T cells were sorted from PBMCs of 4 healthy donors and 2 cancer patients using magnetic bead sorting and used for ex vivo IFN γ ELISPOT. The purity of memory T cell isolation was confirmed by flow cytometric analysis for CD4+ CD45RO + and CD8+ CD45RO + T cells and was determined to be > 95%. The inventors found CD4+ memory responses against ArgLong2 peptide in 2 cancer patients and 3 healthy donors (see fig. 4B). Clear CD8+ memory responses were detectable in 2 healthy donors (see fig. 4C).

IL-4 upregulated arginase 1 expression increases T cell response to ArgLong2

Since Arg 1-specific CD4+ and CD8+ memory T cells were present in cancer patients and healthy donors, it was tested whether these T cells could participate in immune regulation in response to upregulation of Arg1 expression. Arg1 has been previously described as being upregulated in bone marrow cells in response to IL-4.

First, PBMCs from 1 melanoma patient were tested, which had previously been shown to have a strong spontaneous ex vivo response to ArgLong2 peptide. PBMCs were thawed and stimulated with IL-4(50U/ml or 100U/ml), IL-2(120U/ml) or a combination of IL-4 and IL-2 (50U/ml and 60U/ml, respectively) for one week before being tested for response to ArgLong2 in IFN γ ELISPOT. Unstimulated cells were used as controls. A strong significant response was observed in all IL-4 stimulated groups: the highest response was observed for the IL-4 stimulated group compared to stimulation without stimulation or with IL-2 alone, indicating that Arg 1-specific T cells were activated in response to up-regulated Arg1 expression. See fig. 5A.

PBMCs from 2 cancer patients (BC30 (breast cancer), MM27 (malignant melanoma)) and 6 healthy donors, which had previously shown a strong spontaneous ex vivo response to ArgLong2, were then stimulated with low doses of IL-2(120U/ml) or IL-4(100U/ml) for 7 days. Unstimulated cells were used as controls. After 7 days of culture, PBMC responses to ArgLong2 were tested in IFN γ ELISPOT. In IL-4 stimulated cultures, 6 out of 8 cultures showed an increased response to ArgLong2 compared to unstimulated and IL-2 stimulated controls, further confirming a possible general mechanism of Arg1 specific T cell activation in vivo. See fig. 5B.

Discussion of the related Art

The presence of Arg 1-specific T cells has been described previously. Due to its role in targeting immunomodulatory proteins, such T cells may be referred to as "anti-Tregs". anti-Treg responses against other immunomodulatory proteins (e.g., PD-L1 and IDO) are also described. The above experiments indicate that arginase 1-specific anti-tregs are not only present spontaneously in cancer patients and healthy donors, but also as part of the memory T cell bank, as responses to ArgLong2 peptide were observed in isolated CD4+ and CD8+ memory T cell populations.

To maintain immune balance, immune cells (e.g., tregs), different dendritic cell subtypes, myeloid-derived suppressor cells, and M2 macrophages are modulated to suppress or terminate the immune response. The regulatory arm (arm) ensures no response or tolerance to autoantigens. Modulating immune cells suppresses immunity through a number of different cellular and extracellular factors. In contrast, specific anti-Tregs that recognize HLA-restricted derived epitopes generated by intracellular degradation antigens are able to directly eliminate regulatory immune cells. In addition, anti-Tregs can promote local immune activation by secreting effector cytokines.

T_H2-driven lung inflammation increases arginase 1 expression in bone marrow cells. In particular, IL-4, a typical inducer of the macrophage M2 phenotype, induces arginase 1 upregulation. Arginase 1-specific T cells have been shown herein to respond to T _H2 cytokine IL-4. Thus, this indicates arginase 1-specific T _H1 cells infiltrate the increased environment of IL-4 and drive the immune response back to T _H1 pathway, which may be an important role for arginase 1-specific anti-tregs in controlling immunosuppression and promoting inflammation. However, the inhibitory effect of the regulatory immune cells expressing arginase 1 prevents arginase 1 from specifically targeting tregs per se. Thus, in an immune regulatory network, arginase 1-specific anti-Treg inhibits arginase 1+ to regulate immune cell function and vice versa. Thus, under normal physiological conditions, a balance between immune activation and immune suppression may be necessary to maintain immune homeostasis.

Memory T cells specific for arginase 1, which expanded in response to IL-4, were found in healthy individuals, consistent with previous findings for IDO and PD-L1 specific anti-tregs. Circulating IDO-or PD-L1-specific anti-Tregs have been identified in healthy donors, although their detection is less frequent than in cancer patients. In addition, it has been found that the pro-inflammatory cytokines IL-2 and IFN- γ known to induce IDO and PD-L1 expand IDO-or PD-L1-specific T cell populations in human PBMC without additional stimulation.

Immunosuppressive bone marrow cells expressing arginase 1 in the tumor microenvironment suppress anti-tumor T cell responses through L-arginine depletion. In the arginase 1 positive environment, T cells are unable to proliferate, and therefore a promising therapeutic strategy is to reconstitute the adaptive immune response by activating pro-inflammatory arginase 1-specific T cells. Activation of arginase 1-specific anti-Tregs by peptide immunization provides a novel approach to targeting specific immunosuppressive mechanisms in cancer.

When conventional inhibition mechanisms are targeted, the development of new immunotherapeutic approaches in cancer therapy may be the most effective and versatile. As shown in these experiments. Arginase 1-specific anti-tregs occur as a natural part of the immune system and can be readily used to balance away from immunosuppression in cancer.

The peptide ArgLong2, covering the major part of the previously described hot spot region of arginase 1, has proven to be particularly effective in stimulating arginase-specific CD4+ and CD8+ T cell responses, compared to similar peptides of different lengths. Therefore, given its potential to stimulate arginase 1-specific anti-tregs, which exert effector and accessory functions in the tumor microenvironment, ArgLong2 has a particularly high probability of success in the context of immunization.

Example 2 design of additional peptides

To design peptides suitable for mouse immunization experiments and also functional in humans, the sequence of murine arginase 1(SEQ ID NO:11) was compared to the human arginase 1 sequence SEQ ID NO:10 comparison. The level of similarity was particularly high in the hotspot region of human arginase 1 corresponding to positions 161-210 of SEQ ID NO 1. The alignment of this region with the corresponding region in murine arginase 1 is also shown below:

hArgl:

GFSWVTPCISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGK(SEQ ID NO:12)

mArgl:

GFSWVTPCISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKLGIGK(SEQ ID NO:13)

only 2 of the 50 residues are different and are shown in bold and underlined. Leucine in the human sequence is replaced by the highly similar aliphatic amino acid isoleucine in mice and arginine in the human sequence is replaced by the similar basic lysine in mice. Both changes are conservative. Thus, the hot spots are highly conserved between humans and mice. Thus, the polypeptides of the invention include polypeptides consisting of the specific human sequences of the hotspot regions, but in which substitutions have been made to the corresponding mouse sequences. For example, SEQ ID NO 2 corresponds to SEQ ID NO 1, but both have the above-mentioned leucine-isoleucine and arginine-lysine substitutions. The polypeptide consisting of SEQ ID NO. 2 may be referred to herein as mArg Long 2. Similarly, SEQ ID NO 4 corresponds to SEQ ID NO 3, but with the leucine-isoleucine substitution mentioned above. The polypeptide consisting of SEQ ID NO:2 may be referred to herein as mArg Long 3. Similarly, SEQ ID NO 6 corresponds to SEQ ID NO 5, but with the leucine-isoleucine and arginine-lysine substitutions described above. The polypeptide consisting of SEQ ID NO 6 may be referred to herein as mArg Long. Murine peptides are expected to have similar potential as vaccines to their human counterparts.

Example 3 in vivo experiments with the human ArgEong2 peptide in a mouse tumor model

To demonstrate the therapeutic potential of immunization with the ArgLong2 peptide, a mouse model was developed. Since the corresponding ArgLong2 sequence differs in mice by only two amino acids, the human ArgLong2 sequence was used in subsequent experiments. Comparison of the human and mouse ArgLong2 sequences is shown below:

human ArgLong2(SEQ ID NO:1)

-ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRL

Mouse ArgLong2(SEQ ID NO:2)

-ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKL

Materials and methods

Peptide immunization of C57BL/6 mice

Subcutaneous inoculation of animals dissolved in DMSO/H ₂100 μ g peptide in O, which is a 1:1 emulsion containing incomplete Montanide ISA 51 VG. Montanide ISA 51VG + DMSO/H₂O was used as a control vaccine. To subsequently analyze the immune response against the ArgLong2 peptide, mice were sacrificed on day 7 and spleens and draining lymph nodes (dlns) were collected for ELISPOT.

ELISPOT assay of peptide-specific responses

An ELISPOT assay was performed on murine immune cells. Single cell suspensions were prepared from spleens or dLNs by flow through cell filters. After lysing the blood cells, 0.6-0.8X 10⁶Cells/well were seeded onto ELISPOT plates coated with anti-IFN γ antibody. The peptide of interest was added to the indicated wells and cells were incubated with the peptide overnight. The next day, cells were removed, plates were washed and incubated with biotinylated monitor antibody. Finally, visible spots appeared after addition of streptavidin-ALP and substrate. Each spot corresponds to a single IFN γ -producing cell. The plates were analyzed in an Immunospot analyzer and plotted.

Tumor immunization of 15-16/17-20 week old female C57BL/6 mice, 15 mice per group

Each animal was inoculated subcutaneously with 0.5X 10 in 100. mu.l of medium⁶Syngeneic tumor cells to the right flank. An isogenic B16F10 melanoma cell line and an isogenic MC38 colon adenocarcinoma cell line were used. Immunization was started on day 0 and repeated weekly (day 7 and 14). Peptide and control vaccinations were performed as described above.

Tumor growth was monitored and tumors were measured approximately every other day. Tumor volume in V [ mm ]³]＝L×W²Calculated as/2 (where L is the longest diameter and W is perpendicular to L). Before euthanasia, the maximum tumor size of the mice was 864mm³(L＝12mm、W＝12mm)。

Results

ArgLong2 was found to be strongly immunogenic in C57BL/6 mice, showing a high frequency of vaccine-specific T cell responses detected only after one immunization (fig. 6).

In tumor studies, immunization with ArgLong2 significantly delayed tumor growth compared to control immunization. A delay in tumor growth was observed in both the MC38 model of colon adenocarcinoma (fig. 7) and the B16 model of melanoma (fig. 8).

Example 4ArgLong 2-specific T cell clones recognize THP-1 cells expressing arginase 1

Materials and methods

Generation of ArgLong 2-specific T cell clones

Following a standard Rapid Expansion Protocol (REP), T cell cultures enriched for ArgLong 2-specific cells produced ArgLong2 clone cultures: ArgLong 2-specific cells were isolated by magnetic bead sorting TNF α -producing T cells and cloned at-1 cell/well in 96-well plates for limiting dilution. Each well contained 200. mu.l of a mixture of REP (irradiated feeder cells from 3 different buffy coat, alpha CD3 antibody and 6000U/ml IL-2) to promote the growth of ArgLong2 specific T cells.

Quantitation of arginase 1mRNA in THP-1 cells by qPCR

THP-1 cells were stimulated with 20ng/ml IL-13(Trichem) for 48 hours. Cells were harvested, washed in PBS, and pelleted by centrifugation at 300g for 5 minutes. The pellet was kept on ice and resuspended in 350. mu.l of RLT Plus-buffer (Qiagen). RNA was purified using RNAeasy kit (Qiagen) according to the manufacturer's instructions and finally eluted with 30. mu.l of RNA-free water. The RNA concentration was measured on a NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA was stored at-80 ℃.

Total RNA was reverse transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems). For each reaction, 1000ng of RNA was reverse transcribed. For RT-qPCR, the cDNA was diluted 1:5 and analyzed by RT-qPCR using TaqMan gene expression assay on a Roche Lightcycler 480 instrument. RT-qPCR was performed in quadruplicate and the data was analyzed using ddCT method, normalized to the expression level of the housekeeping gene RPLPO and control samples. For the unamplified low concentration samples, Ct was set to 40. The no reverse transcriptase control (cDNA reaction set up without reverse transcriptase) was used as a control for specific amplification. The Arg1 human primer was purchased from Thermo fisher (unpublished sequence, product number Hs00163660_ m 1).

Determination of THP-1 cell recognition by ArgLong 2-specific T cells

Untreated and IL-4/IL-13 treated THP-1 cells were washed and divided into two groups: HLA-DR/DQ/DP blocking antibody (clone Tu 39) was added to the first panel at a concentration of 10. mu.g/ml for 20min at 37 ℃. The THP-1 cells of the second group were untreated. After 20min, HLA-II blocked THP-1 cells were washed. THP-1 cells treated with IL-4/IL-13 (with or without HLA-II blockade) were then mixed with ArgLong2 peptide specific T cells at an effective target ratio of 2:1 and then treated according to standard intracellular cytokine staining protocols.

Results

To further assess the function of ArgLong 2-specific T cells, T cell clones were generated from healthy donors previously identified as strong ArgLong2 responses, and tested for their ability to specifically recognize the target expressing arginase 1.

Th2 cytokines (e.g., IL-4 or IL-3) are known to upregulate the expression of ARG1 in bone marrow cells. To test whether ArgLong 2-specific T cells would specifically recognize Th2 cytokine-treated bone marrow cells, ArgLong2T cell clones were tested for their ability to recognize HLA-matched monocytic THP-1 cell lines pre-stimulated with IL-4(100U/ml) or IL-13(20U/ml) for 48 hours.

Th 2-cytokine treated THP-1 showed increased arginase 1 expression. In this regard, FIG. 9C shows that arginase 1 expression is induced in THP-1 cells when pretreated with IL-13, thereby increasing presentation of arginase 1-derived peptide epitopes on the surface of these cells.

ArgLong 2-specific CD4T cell clones were demonstrated to recognize IL-4 and IL-13 treated THP-1 cells as indicated by increased production of TNF α and IFN γ to these cells compared to untreated THP-1 (FIG. 9A). Blockade of HLA class II molecules abrogated increased recognition of IL-4 or IL-13 treated THP-1 cells, indicating that increased recognition of IL-4 and IL-13 treated THP-1 cells is dependent on HLA class II presentation of ARG1 peptides. FIG. 9B illustrates, in dot-plot form, the representative data of FIG. 9A, showing the production of IFN γ and TNF α in response to IL-4 and IL-13 treated THP-1 cells with or without HLA class II blockade.

In a separate experiment, PBMCs from healthy donors (HD22, known to have a pre-existing ex vivo response to ArgLong2) were treated with IL-4(100U/ml) or IL-13(20U/ml) for 7 days to increase the presentation of arginase 1-derived peptides in PBMC cultures, thereby stimulating the intrinsic ArgLong 2-specific T cells present in PBMCs. PBMC were analyzed in an IFN γ ELISPOT assay 7 days after stimulation with IL-4 or IL-13 to examine changes in the frequency of ArgLong 2-specific T cells.

An increased ArgLong2 response was observed in IL-4 and IL-13 treated PBMC cultures compared to untreated controls. The magnitude of the IL-13 stimulated ArgLong2 response was comparable to that observed after in vitro ArgLong2 peptide stimulation, indicating a strong activation of arginase 1-specific cells under Th2 cytokine conditions.

Sequence of

It is intended to indicate a sequence derived from murine arginase 1, which contains at least one difference with respect to the corresponding region of human arginase 1. Residues that are not identical to the corresponding human sequence are bold and underlined. Murine and human arginase 1 are the same length and therefore start and stop positions are the same.

full-Length human arginase 1 (NP-000036.2) (SEQ ID NO:10)

MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQL AGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGV IWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVP GFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSM TEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSF TPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNP SLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK

The regions identified as immunogenic hotspots are indicated in bold and underlined

full-Length murine arginase 1 (NP-031508.1) (SEQ ID NO:11)

MSSKPKSLEI IGAPFSKGQP RGGVEKGPAA LRKAGLLEKL KETEYDVRDH GDLAFVDVPN DSSFQIVKNP RSVGKANEEL AGVVAEVQKN GRVSVVLGGD HSLAVGSISG HARVHPDLCV IWVDAHTDIN TPLTTSSGNL HGQPVSFLLK ELKGKFPDVP GFSWVTPCIS AKDIVYIGLR DVDPGEHYII KTLGIKYFSM TEVDKLGIGK VMEETFSYLL GRKKRPIHLS FDVDGLDPAF TPATGTPVLG GLSYREGLYI TEEIYKTGLL SGLDIMEVNP TLGKTAEEVK STVNTAVALT LACFGTQREG NHKPGTDYLK PPK

The regions identified as immunogenic hotspots are indicated in bold and underlined

Hot spot in human arginase 1-position 161-210 of SEQ ID NO:10

GFSWVTPCISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGK(SEQ ID NO:12)

Hot spot in murine arginase 1-position 161-210 of SEQ ID NO:11

GFSWVTPCISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKLGIGK(SEQ ID NO:13)

Residues that are not identical to the corresponding human sequence are bold and underlined.

Sequence listing

<110> IO Biotech ApS

<120> arginase 1 polypeptides

<130> N414498WO

<150> GB1815549.9

<151> 2018-09-24

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1 (ArgLong2)

<400> 1

Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro

1 5 10 15

Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met

20 25 30

Thr Glu Val Asp Arg Leu

35

<210> 2

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of murine arginase 1 (mArg Long2)

<400> 2

Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro

1 5 10 15

Gly Glu His Tyr Ile Ile Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met

20 25 30

Thr Glu Val Asp Lys Leu

35

<210> 3

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1 (ArgLong3)

<400> 3

Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro

1 5 10 15

Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met

20 25 30

<210> 4

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of murine arginase 1 (mArg Long3)

<400> 4

Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro

1 5 10 15

Gly Glu His Tyr Ile Ile Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met

20 25 30

<210> 5

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1 (ArgLong)

<400> 5

Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro

1 5 10 15

Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met

20 25 30

Thr Glu Val Asp Arg Leu Gly Ile Gly Lys

35 40

<210> 6

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of murine arginase 1 (mArg Long)

<400> 6

Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro

1 5 10 15

Gly Glu His Tyr Ile Ile Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met

20 25 30

Thr Glu Val Asp Lys Leu Gly Ile Gly Lys

35 40

<210> 7

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1 (Arg1-18)

<400> 7

Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu

1 5 10 15

His Tyr Ile Leu

20

<210> 8

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1 (Arg1-19)

<400> 8

Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys

1 5 10 15

Tyr Phe Ser Met

20

<210> 9

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1 (Arg1-20)

<400> 9

Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu

1 5 10 15

Gly Ile Gly Lys

20

<210> 10

<211> 322

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 10

Met Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser

1 5 10 15

Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg

20 25 30

Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys

35 40 45

Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe

50 55 60

Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu

65 70 75 80

Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val

85 90 95

Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala

100 105 110

Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp

115 120 125

Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro

130 135 140

Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro

145 150 155 160

Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr

165 170 175

Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr

180 185 190

Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile

195 200 205

Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys

210 215 220

Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe

225 230 235 240

Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu

245 250 255

Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly

260 265 270

Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu

275 280 285

Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe

290 295 300

Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro

305 310 315 320

Pro Lys

<210> 11

<211> 323

<212> PRT

<213> mouse (Mus musculus)

<400> 11

Met Ser Ser Lys Pro Lys Ser Leu Glu Ile Ile Gly Ala Pro Phe Ser

1 5 10 15

Lys Gly Gln Pro Arg Gly Gly Val Glu Lys Gly Pro Ala Ala Leu Arg

20 25 30

Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Thr Glu Tyr Asp Val Arg

35 40 45

Asp His Gly Asp Leu Ala Phe Val Asp Val Pro Asn Asp Ser Ser Phe

50 55 60

Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Asn Glu Glu Leu

65 70 75 80

Ala Gly Val Val Ala Glu Val Gln Lys Asn Gly Arg Val Ser Val Val

85 90 95

Leu Gly Gly Asp His Ser Leu Ala Val Gly Ser Ile Ser Gly His Ala

100 105 110

Arg Val His Pro Asp Leu Cys Val Ile Trp Val Asp Ala His Thr Asp

115 120 125

Ile Asn Thr Pro Leu Thr Thr Ser Ser Gly Asn Leu His Gly Gln Pro

130 135 140

Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Phe Pro Asp Val Pro

145 150 155 160

Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr

165 170 175

Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Ile Lys Thr

180 185 190

Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Lys Leu Gly Ile

195 200 205

Gly Lys Val Met Glu Glu Thr Phe Ser Tyr Leu Leu Gly Arg Lys Lys

210 215 220

Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ala Phe

225 230 235 240

Thr Pro Ala Thr Gly Thr Pro Val Leu Gly Gly Leu Ser Tyr Arg Glu

245 250 255

Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly

260 265 270

Leu Asp Ile Met Glu Val Asn Pro Thr Leu Gly Lys Thr Ala Glu Glu

275 280 285

Val Lys Ser Thr Val Asn Thr Ala Val Ala Leu Thr Leu Ala Cys Phe

290 295 300

Gly Thr Gln Arg Glu Gly Asn His Lys Pro Gly Thr Asp Tyr Leu Lys

305 310 315 320

Pro Pro Lys

<210> 12

<211> 50

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of human arginase 1

<400> 12

Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr

1 5 10 15

Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr

20 25 30

Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile

35 40 45

Gly Lys

50

<210> 13

<211> 50

<212> PRT

<213> Artificial sequence

<220>

<223> peptide fragment of murine arginase 1

<400> 13

Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr

1 5 10 15

Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Ile Lys Thr

20 25 30

Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Lys Leu Gly Ile

35 40 45

Gly Lys

50

Claims (12)

1. A polypeptide consisting of any one of the following amino acid sequences:

a.ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRL(SEQ ID NO:1)；

b.ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKL(SEQ ID NO:2)；

c.ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSM(SEQ ID NO:3)；

d.ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSM(SEQ ID NO:4)；

e.ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGK(SEQ ID NO:5)；

f.ISAKDIVYIGLRDVDPGEHYIIKTLGIKYFSMTEVDKLGIGK(SEQ ID NO:6)；

g. a sequence according to any one of a. -f., wherein up to three amino acids are replaced by conservative substitutions, provided that the polypeptide does not exhibit significantly reduced immunogenicity to arginase 1 as compared to a polypeptide consisting of the corresponding sequence without the substitutions; or

h. A polynucleotide encoding a polypeptide of any one of a.

2. The polypeptide of claim 1, consisting of the amino acid sequence of SEQ ID NO 1.

3. The polypeptide of claim 1 or 2, wherein the C-terminal amino acid is replaced by a corresponding amide.

4. A composition comprising the polypeptide or polynucleotide of any one of claims 1 to 3 and an adjuvant.

5. A composition according to claim 4, comprising at least one pharmaceutically acceptable diluent, carrier or preservative.

6. The composition according to claim 4 or 5, wherein the adjuvant is selected from the group consisting of: bacterial DNA-based adjuvants, oil/surfactant-based adjuvants, viral dsRNA-based adjuvants, imidazochiniline and Montanide ISA adjuvants.

7. A method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a polypeptide or polynucleotide of any one of claims 1 to 3 or a composition of claims 4 to 6.

8. The method of claim 7, wherein the disease or disorder is characterized at least in part by inappropriate or excessive immunosuppressive function of arginase, and/or wherein the disease or disorder is cancer.

9. The method of claim 7 or 8, wherein the disease or disorder is cancer, and optionally wherein the method further comprises the simultaneous or sequential administration of an additional cancer therapy, preferably an antibody.

10. The method of any one of claims 7 to 9, wherein the cancer is breast, lung, colon or prostate cancer, or is melanoma, or is leukemia, preferably Acute Myeloid Leukemia (AML).

11. A method of stimulating arginase 1 specific T cells, comprising contacting said cells with the polypeptide of any one of claims 1 to 3 or the composition of claims 4 to 6.

12. The method of claim 11, wherein the cells are present in a sample taken from a healthy subject or from a cancer patient, optionally a tumor sample.

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